JP2001517679A - Method for purifying antithrombin III - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for purifying antithrombin III from a starting material containing the complex by adsorbing a heparin / AT III or heparinoid / AT III complex to an anion exchange material and then cleaving the eluting AT III. On how to do it.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel method for purifying antithrombin III.

[0002] Antithrombin III (AT III) comprises thrombin, IXa, Xa, XIa
A plasma protein that acts as a coagulation inhibitor by inhibiting factor XIIa.

[0003] AT III deficiency or hereditary thrombotic propensity is an autosomal dominantly inherited disorder that has a propensity for thrombosis and embolism due to reduced AT III formation.

[0004] Acquired AT III deficiency can occur, for example, with disseminated intravascular coagulation (DIC), sepsis, cirrhosis or nephrotic syndrome.

[0005] Symptoms of AT III deficiency can occur in the case of heart valve prostheses, postoperative thromboembolic complications, in estrogen therapy, or even in asparaginase therapy.

Because of the high affinity of AT III for heparin and heparinoids, it is necessary to separate heparin or heparinoids when producing pure antithrombin III preparations.

According to EP 0 307 002 A1, the cleavage of this AT III / heparin or AT III / heparinoid complex is carried out by immobilized protamine, and heparin is bound to the immobilized protamine, ATIII is recovered from the supernatant. Prior to treatment with immobilized protamine, the AT III / heparin or AT III / heparinoid conjugates are each adsorbed to an ion exchange material using saline at pH 7.5 and eluted from unwanted proteins. Purify. The complex was not cleaved by ion exchange chromatography and undesired accompanying protein separation was achieved, indicating that such complexes themselves remained adsorbed unchanged.

[0008] A further possible method for purifying AT III is by affinity chromatography using heparin-Sepharose.

For example, Prep. Biochem. 13 (1) (1983), pp. 1-20, describes a method for purifying ATIII. First, ATIII was purified using heparin sepharose. And purified by affinity chromatography, followed by ion exchange chromatography and gel chromatography. AT III elutes from heparin-Sepharose at pH 7.4. Ion exchange chromatography was performed on DEAE-Sepharose, with AT III bound at pH 8.6.

[0010] Thrombosis Research 5 (1974), pp. 431-452, also describes the purification of ATIII using heparin-Sepharose, and the adsorption is carried out at pH 8.5 by desorption, ie, immobilized heparin. Cleavage of the complex between ATIII and ATIII occurs at pH 7.5. Subsequent ion exchange chromatography using DEAE-Sephadex revealed that AT III bound at pH 8.0 and eluted at pH 7.4.

A similar method is described in DE 2 243 688. This publication also discloses the purification of ATIII on a bridged heparin-agarose gel, the adsorption to heparin gel at pH 8.5 and the desorption, ie the cleavage of ATIII from the immobilized heparin, at pH7. .3.

The present invention provides a method for producing antithrombin III preparations in high purity and high yield by allowing AT III to be recovered with as little as possible heparin and heparinoid respectively. , Based on the goal of providing new ways.

According to the present invention, the object is to provide a process for purifying AT III from a starting material comprising AT III / heparin or AT III / heparinoid complex, comprising AT III / heparin or AT III. / Heparinoid complex is adsorbed to an anion exchange material, and then the ATIII is cleaved and eluted from the adsorbed complex. This cleavage leaves heparin on the anion exchange material. That is, selective elution of AT III occurs.

[0014] Preferably, the cleavage according to the invention is carried out with a buffer in the range pH 8.5 to 10.5.

Surprisingly, cleavage of the heparin / AT III or heparinoid / AT III complex, respectively, is possible in the course of anion exchange chromatography,
This has been found to be possible even at pH higher than 8.5.

From the anion exchange material, AT III can be selectively eluted with respect to heparin, ie, the binding of heparin to the adsorbent, which is not a covalent bond in contrast to heparin-sepharose, remains intact. It was found that the complex formation between AT III and heparin or heparinoid could be cleaved, respectively. In the prior art, it was originally assumed that AT III had a very high affinity for heparin at such pH (see Thrombosis Research (1974) or DE 2243 688), and even for covalently bound heparin, Cleavage of the complex (ie, cleavage of AT III from immobilized heparin) was always carried out at low pH values, but from adsorbed AT III / heparin or AT II // heparinoid complex. It was found that the selective elution of AT III was possible by anion exchange chromatography.

However, it has been found within the scope of the present invention that, even at very high pH values, cleavage of the complex can be achieved while heparin remains bound to the anion exchanger.

Preferably, in the method according to the invention, elution is carried out with a buffer having a conductivity of 15 to 50 mS. Optimal selectivity elution is achieved at such conductivity. That is, the conductivity is one that is sufficiently high to allow substantially complete cleavage of the complex, but not so high that heparin or heparinoid respectively elute together. The conditions for adsorption and desorption respectively generally depend on the anion exchange material used,
There is a substantial correlation between the conductivity and the pH value of the buffer.

In particular, the interdependence between pH and conductivity is such that at low conductivity, for example around 20 mS, the pH of the buffer solution is also low, for example 8.5, and vice versa. is there. As a buffer solution, for example, a solution containing Tris, phosphate, or glycine is used.

Preferably, prior to the adsorption, heparin or heparinoid, respectively, is 30 to 3
Mix in an amount of 000 U / mL. This results in all AT III in the starting material
Is present as a complex, eliminating loss of yield due to the presence of free ATIII.

Preferably, the method according to the invention is carried out in a two-step chromatographic purification,
In the first step, the complex is adsorbed on the anion exchange material, and the stable complex is eluted in the pH range of 6.0 to 7.5. Next, the complex of the present invention is adsorbed, and AT III is cleaved or eluted from the complex. If the pH is between 8.5 and 10.5, the buffer is preferably brought to a high conductivity during elution, for example between 10 and 60 mS, preferably between 15 and 5 mS.
Adjust to 0 mS, most preferably 20 to 35 mS.

Since AT III is preferably used as a therapeutic agent, it is often necessary to treat it for virus inactivation. This treatment is preferably carried out at the stage of the AT III / heparin or AT III / heparinoid complex, since AT III is more stable in complex form than in free form. Preferably, the treatment of virus inactivation is also performed in two steps, ie in two independent virus inactivation methods.

This inactivation treatment is preferably by a surfactant and / or a heat treatment, for example a heat treatment in the solid state, in particular a steam treatment as described in EP-0 159 311, EP-0 519 901 or EP-0 674 531. to be certain.

Methods for inactivating the virus further include chemical or chemical / physical methods, such as the chaotropic substances described in WO 94/13329, DE 44 34 538 or EP-0 13 1 740 (solvent). Alternatively, treatment by light inactivation is included.

[0025] Nanofiltration is also a preferred method for reducing virus within the scope of the present invention.

As anion exchange substances, in principle, all anion exchange substances having an affinity for heparin or heparinoid can be used. For example, a cellulose-based anion exchange material having diethylaminoethyl groups (DEAE-Sephacel ™, DE32, DE52, etc., or Express Ion D (all from Whatman)) or CH ₂ N ⁺ (CH ₃ ) ₃ groups Cellulose-based anion exchange material (QA52 or Express Ion Q (from Whatman)), anion exchange material based on cross-linked dextran with diethylaminoethyl groups (DEAE-Sephadex ™), agarose with diethylaminoethyl groups Base anion exchange material (DEAE-Sepharose CL6B (trademark), DEAE-Sepharose Fast Flow ™), diethyl- [2
Anion exchange material based on cross-linked dextran having -hydroxypropyl] -aminoethyl groups (QAE-Sephadex ™), anion exchange material based on agarose having CH ₂ N ⁺ (CH ₃ ) ₃ groups (Q- Sepharose Fas
t Flow ™, Q-Sepharose High Performance ™, Q-Sepharose Big Beads ™) or an anion exchange material based on a copolymer of agarose and dextran (Q-Sepharose XL) (all from Pharmacia), function N-acryloyl-2 as a neutral anion exchange material
-Spherical chromatographic gel (DEAE-Tris-Acryl (TM)) produced by a copolymerization reaction of -amino-2-hydroxymethyl-1,3-propanediol with an anionic acrylic derivative having a diethylaminoethyl group, crosslinked A non-compressible silica-dextran matrix with reactive diethylaminoethyl anion exchange groups (DEAE-Spherodex ™), embedded with porous silica gel within a coated dextran matrix, made of strong polystyrene particles. , A gel filled with pores with a hydrogel having quaternary amine groups having strong anion exchange activity (Q-Hyper-D ™) (all Sepraco
r); a hydrophilic surface having strong macropores having N ⁺ (C ₂ H ₅ ) ₂ or N ⁺ (CH ₃ ) ₃ groups (Macroprep DEAE ™, Macroprep
Q ™ (all from BioRad)); diethylaminoethyl-diethyl- (
Anion exchange materials having 2-hydroxypropyl) -aminoethyl and CH ₂ N ⁺ (CH ₃ ) ₃ groups (DEAE-Toyopearl ™, QAE-Toyopearl ™, Toyopearl Super-Q ™ (all manufactured by Tosohass )), An anion exchange resin consisting of a porous polymethacrylate / polyacrylate gel (Protein PAK DEAE ™ (Waters)); hydrophilicity consisting of oligoethylene glycol-dimethyl acrylate, glycidyl-methacrylate and pentaerythritol dimethyl acrylate Anion exchange materials based on copolymers with a surface (Fractogel EMD-TMAE ™, Fractogel EM
D-DEAE ™, Fractogel EMD-DMAE ™, an anion exchange resin based on silica gel made of pressure-resistant, spherical, porous chromatography particles (Microsphere 1000 TMAE ™, Licrosp.
her 1000 DEAE (TM), Microsphere 4000 DMAE
(Trademark) (all made by MERCK)).

In a particularly preferred embodiment of the method according to the invention, the human plasma or the plasma fraction containing AT III is mixed with heparin or heparinoid to form an AT III / heparin or AT III / heparinoid complex, The conjugate is subjected to a heat treatment to inactivate the infectious agent. This treatment is preferably carried out in the presence of a stabilized organic polyvalent salt, such as citrate and / or ammonium sulphate, preferably from 40 to 40%.
The treatment is carried out at a temperature of 70 ° C. for 3 to 30 hours, and a treatment at a temperature around 60 ° C. for about 10 hours is particularly preferred.

In the method according to the invention, preferably the starting material is human plasma or a (human) plasma fraction containing AT III, preferably cryoprecipitate-depleted plasma or Cohn fraction, preferably Cohn fraction IV.

The present invention will be described in more detail with reference to the following examples, but is not limited thereto.

Example 1 93.2 L of cryoprecipitate-removed plasma was mixed with 7.5 × 10 ⁶ U of heparin, stirred for 1/2 hour, and mixed with 1 g of DEAE-Sephadex A50 (Pharmacia) per liter. did. The gel was separated and unbound proteins were removed with a citrate buffered NaCl solution (1 g / L NaCl, pH 7.5) and AT II
The I / heparin complex was recovered by elution with a buffer having a conductivity of 44 mS and a pH of 7.5. This solution was then heated to 60 ° C. for 10 hours in the presence of a stabilized organic polyvalent salt (160 g / L Na-citric acid) to inactivate any pathogenic microorganisms that may be present.

[0031] The precipitate formed was separated by centrifugation or filtration and discarded. The clear solution was buffered again to pH 9.0 and conductivity 12.2 mS. The AT III / heparin conjugate was bound to a chromatography column consisting of 1000 mL of Q-Sepharose Fast Flow ™ (Pharmacia), and the AT III was selectively selected with a buffer having a pH of 9.0 and a conductivity of 26 mS. Eluted.

[Table 1]

Example 2: (Best embodiment of the present invention as considered by the applicant at present) After removing the cryoprecipitate and prothrombin complex, the plasma 24
0.5 L was mixed with 1.85 × 10 ⁶ U of heparin. The ATIII / heparin complex was adsorbed using 15 g of DE52 cellulose (manufactured by Whatman) per liter, and the product was recovered by elution with a buffer having a conductivity of 45 mS and a pH of 8.0. After pasteurization (60 ° C., 10 hours), the solution was re-buffered and treated in US Pat. No. 4,540,57.
According to No. 3, at 25 ° C., Triton ™ × 100 (polyethylene glycol-tert-octylphenyl ether, Tween ™ 80 (polyoxyethylene sorbitan-mono-oleate) and tri- (n-butyl) phosphate ( TNBP).

AT III was chromatographed on a 25 mL Q-Sepharose Fast Flow ™ column (Pharmacia), pH 9.8, conductivity 2
Recovered by elution with 3mS buffer. By ultracentrifugation and diafiltration,
After adjusting AT III to 100 U / mL, the solution was put into a container by filtration under aseptic conditions, and freeze-dried if desired. Results after lyophilization AT III 96 U / mL heparin 0.8 U / mL AT III / protein 6.2 U / mL Triton, Tween, TNBP <Detection limit Heparin binding of AT III> 95% *) *) According to European Pharmacopoeia

Example 3: Toyopearl ™ Q-650Th ™ (T
Example 2 was repeated with the aid of (iso Haas). Result (eluate) AT III 4.5 U / mL heparin 0.1 U / mL AT III / protein 3.2 U / mL

Example 4 Example 2 was repeated using Express Ion Q (Whatman) instead of Q-Sepharose. Result (eluate) AT III 8.0 U / mL Heparin 0.8 U / mL AT III / protein 5.5 U / mL

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP , KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor: Jilvia Nepple Austria, A-1030 Wien, Ludengasse 19/15 ( 72) Inventor Wolfgang Schoenhofer, Aer-3100 Sankt-Pelten, Austria, Ringernutgasse 5 / Tze No. 1 (72) Inventor Hans-Peter Schwarz, Austria, A-1180 Wien, Schindragasse 32

Claims (8)

[Claims] 1. Starting materials containing antithrombin III / heparin or antithrombin III / heparinoid complexes are prepared from antithrombin I / heparin, respectively.
A method for purifying II, wherein the complex is adsorbed on an anion exchange material, and antithrombin III is separated from the complex by elution. 2. The method according to claim 1, wherein the elution is performed with a buffer having a pH in the range of 8.5 to 10.5. 3. The buffer for elution is 10 to 60 mS, preferably 15 to 50 mS.
Method according to claim 1 or 2, characterized in that it has a conductivity of mS, most preferably 20-35 mS. 4. A mixture of heparin or heparinoid in an amount of 30 to 3000 U / mL with a starting material containing antithrombin III,
The method according to any one of claims 1 to 3, wherein a complex of II / heparin or antithrombin III / heparinoid is formed. 5. A two-step elution of the antithrombin III / heparin or antithrombin III / heparinoid complex in a first step, in which the complex is adsorbed on an anion exchange material and is eluted in the pH range of 6.0 to 7.5. The method according to any one of claims 1 to 4, wherein the method is subjected to chromatography purification. 6. The method according to claim 1, wherein a virus inactivating step is provided.
5. The method according to any one of 5. 7. The human plasma or antithrombin III-containing plasma fraction is mixed with heparin or heparinoid, and the mixture is mixed with heparin / antithrombin III, respectively.
Alternatively, a heparinoid / antithrombin III complex is formed, which complex is inactivated for inactivation of the infectious agent, optionally in the presence of a stabilized organic polyvalent salt, preferably citrate and / or ammonium sulfate, The method according to any of the preceding claims, characterized in that the heat treatment is carried out at a temperature in the range from 40 to 70C for 3 to 30 hours, preferably at a temperature near 60C for 10 hours. 8. The method according to claim 1, wherein the starting material is human plasma or a plasma fraction containing antithrombin III, preferably cryoprecipitate-depleted plasma.